WO2005097176A2 - Modulation du transport d'oxalate - Google Patents

Modulation du transport d'oxalate Download PDF

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Publication number
WO2005097176A2
WO2005097176A2 PCT/US2005/010227 US2005010227W WO2005097176A2 WO 2005097176 A2 WO2005097176 A2 WO 2005097176A2 US 2005010227 W US2005010227 W US 2005010227W WO 2005097176 A2 WO2005097176 A2 WO 2005097176A2
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Prior art keywords
oxalate
oxalobacter
composition
lysate
enzymes
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PCT/US2005/010227
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English (en)
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WO2005097176A3 (fr
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Marguerite Hatch
Ammon B. Peck
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University Of Florida Research Foundation, Inc.
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Publication of WO2005097176A2 publication Critical patent/WO2005097176A2/fr
Publication of WO2005097176A3 publication Critical patent/WO2005097176A3/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/45—Transferases (2)

Definitions

  • the invention relates to compositions and methods for treatixig animal subjects, including humans, suffering from renal disease with Oxalobacter sp. bacteria or Oxalobacter enzymes.
  • Oxalobacter formigenes maintains an important symbiotic relationship with its hosts by regulating oxalic acid homeostasis.
  • O. formigenes may influence several oxalate-related diseases, since the absence of the bacterium correlates with hyperoxaluria and episodes of kidney stone formation. More notably, however, colonization with O. formigenes significantly lowers hyperoxaluria in rats fed a diet rich in oxalate. Thus, lack of colonization of the GI tract with O. formigenes appears to be an additional risk factor for calcium oxalate urolithiasis.
  • the invention relates to the discovery that the actions of O. formigenes and/or lysate- enzyme preparations thereof promote an enhanced enteric elimination of oxalate. This enteric excretion was notably higher for animals suffering from chronic renal failure than for animals with normal renal function. The administration of O.
  • the oxalate degradation produces an outwardly directed concentration gradient and the active component has an oxalate degrading capacity between about 0.1 ⁇ mole to about 100.0 ⁇ mole oxalate/min/mg protein.
  • the method of increasing the number of oxalate-degrading bacteria or amount of oxalate-degrading bacteria enzymes in the gastrointestinal tract in the animal comprises administering a composition comprising Oxalobacter to the animal subject.
  • the oxalate-degrading bacteria is Oxalobacter formigenes and the bacteria can be viable and/or a lysate thereof.
  • the composition comprises a pharmaceutically acceptable carrier that is resistant to degradation by gastric acidity.
  • a pharmaceutical composition comprises a lysate/enzyme preparation of Oxalobacter bacterium and Oxalobacter enzymes, whereby, the composition is formulated to prevent degradation in acidic conditions.
  • the acidic conditions are the conditions found in the stomach, and the composition can be encapsulated in a capsule which does not dissolve at 37° C at pH ⁇ 6.0.
  • the composition comprises a lysate of substrate-specific Oxalobacter bacteria and oxalate-degrading enzymes.
  • the oxalate- degrading enzymes are preferably formyl-CoA transferase and oxalyl CoA decarboxylase.
  • the composition is formulated to provide an oxalate degrading capacity of between about 50 mg to about 900mg oxalate/day.
  • Each dose of the composition comprises about 1.Omg to about 20 mg of active enzyme component with an oxalate degrading capacity between about 0.1 ⁇ mole to about 100.0 ⁇ mole oxalate/min/mg protein.
  • a method of treating a patient suffering from renal disease comprises administering to the patient a composition comprising oxalate substrate specific Oxalobacter lysates and Oxalobacter enzymes, wherein administration of the composition increases the rate of oxalate transport from a parenteral site to the intestinal lumen.
  • the Oxalobacter lysates and Oxalobacter enzymes are in a ratio of 1 : 1 (v/v) up to a ratio of 1:50 (v/v).
  • the Oxalobacter lysate and Oxalobacter enzyme compositions are formulated to provide an oxalate degrading capacity of between about 50 mg to about 900mg oxalate/day. In accordance with the invention, these ratios can be manipulated according to the patient's response to the treatment. Therefore, in some patients the Oxalobacter lysates and Oxalobacter enzymes are in a ratio of 5:1 (v/v) up to a ratio of 50:1 (v/v).
  • the method further provides administration of oxalate- degrading enzymes, such as, formyl-CoA transferase and oxalyl CoA decarboxylase.
  • oxalate- degrading enzymes such as, formyl-CoA transferase and oxalyl CoA decarboxylase.
  • the enzymes can be administered before, during and/or after administration of the composition comprising Oxalobacter lysates and Oxalobacter enzymes.
  • FIG. 1 is a graph showing a reduction of hyperoxaluria via increased secretion of oxalic acid across the intestinal wall.
  • Grp I non-treated
  • Grp II ethylene glycol (EG) diet
  • Grp III EG diet + O. formigenes
  • Grp IN EG + oxalate diet + O. formigenes.
  • FIG. 2 is a graph showing colonic oxalate transport in hyperoxaluric-induced CRF rats treated with encapsulated Oxalobacter lysate or placebo.
  • FIG. 3 is a graph showing regulation of hyperoxaluria in the rat model via reduction of oxalic acid absorption across the intestinal wall.
  • FIG. 4 is a graph showing colonic oxalate transport in naturally colonized rats with normal renal function.
  • FIG. 3 is a graph showing regulation of hyperoxaluria in the rat model via reduction of oxalic acid absorption across the intestinal wall.
  • FIG. 4 is a graph showing colonic oxalate transport in naturally colonized rats with normal renal function.
  • FIG. 5 is a graph showing that there were no significant effects of the membrane fragment preparation on oxalate transport across the distal colon (n
  • FIG. 7 is a graph showing that similar to normal healthy control rats with both kidneys intact, the distal segment of the UN rats supports a significant basal net absorptive flux of oxalate.
  • FIG. 8 is a graph showing that colonic oxalate transport was significantly altered in the rats receiving the Oxalobacter lysate compared to the placebo-treated rats.
  • the invention provides methods and compositions for increasing the rate of oxalate transport from a parenteral site to the intestinal lumen in an animal subject suffering from renal failure. Studies described herein demonstrate that oxalate excretion into the lumen of the large, intestine, where it can be degraded innocuously by the local microflora, decreases oxalate levels in the urine.
  • This enteric excretion of oxalate that bypasses the kidneys should have a significant impact on reducing hyperoxalemia, hyperoxaluria, oxalosis and the resulting various pathophysiological and debilitating conditions.
  • the methods and compositions of the invention are particularly advantageous for preventing hyperoxaluria and oxalate crystal deposition in the tissue and the formation of kidney stones containing calcium oxalate.
  • ameliorated or “treatment” refers to a symptom which approaches a normalized value (for example a value obtained in a healthy patient or individual), e.g., is less than 50% different from a normalized value, preferably is less than about 25% different from a normalized value, more preferably, is less than 10% different from a normalized value, and still more preferably, is not significantly different from a normalized value as determined using routine statistical tests.
  • a "pharmaceutically acceptable” component is one that is suitable for use with humans and/or animals without undue adverse side effects (such as toxicity, irritation, and allergic response) commensurate with a reasonable benefit/risk ratio.
  • the term "safe and effective amount” refers to the quantity of a component which is sufficient to yield a desired therapeutic response without undue adverse side effects (such as toxicity, irritation, or allergic response) commensurate with a reasonable benefit/risk ratio when used in the manner of this invention.
  • therapeutically effective amount is meant an amount of a compound of the present invention effective to yield the desired therapeutic response.
  • modulate it is meant that any of the mentioned activities, are, e.g., increased, enhanced, agonized (acts as an agonist), promoted, decreased, reduced, suppressed blocked, or antagonized (acts as an agonist).
  • Modulation can increase activity more than 1- fold, 2-fold, 3-fold, 5-fold, 10-fold, 100-fold, etc., over baseline values. Modulation can also decrease its activity below baseline values.
  • the below described preferred embodiments illustrate adaptations of these methods and compositions. Nonetheless, from the description of these embodiments, other aspects of the invention can be made and/or practiced based on the description provided below.
  • the invention provides a method for increasing the rate of oxalate transport from a parenteral site to the intestinal lumen in an animal subject suffering from renal failure.
  • the method includes the steps of providing an animal subject with renal failure and increasing the number of Oxalobacter bacteria or amount of Oxalobacter enzymes in the gastrointestinal tract of the animal.
  • An animal subject suffering from renal failure can be a subject having low levels of Oxalobacter within the intestines or a subject entirely lacking intestinally- residing Oxalobacter. Any suitable method for increasing the number of Oxalobacter bacterium or amount of Oxalobacter enzymes in the gastrointestinal tract in the animal may be used.
  • Oxalobacter that are administered can be in the form of live organisms, as these are non- pathogenic bacteria normally inhabiting the guts of mammals. Portions of oxalate-degrading bacteria can also be administered, such as an Oxalobacter cellular lysate or isolated oxalate- degrading bacterial enzymes. Alternatively, a nucleic acid encoding one or more Oxalobacter enzymes can be administered to the subject. Nucleic acid (i.e., DNA) sequences encoding these enzymes are known to those skilled in the art and are described in, for example, WO 98/16632.
  • the subject invention pertains to the preparation and administration of cells of oxalate-degrading bacteria of the species, Oxalobacter formigenes, to the human or animal intestinal tract where the activity of the microbes reduces the amount of oxalate present in the intestine thereby causing a reduction of concentrations of oxalate in the kidneys and in other cellular fluids.
  • the introduced cells degrade oxalate and replicate in the intestinal habitat so that progeny of the initial cells colonize the intestine and continue to remove oxalate. This activity reduces the risk for formation of kidney stones as well as other disease complications caused by oxalic acid.
  • the specific strains of O In a preferred embodiment for human use, the specific strains of O.
  • formigenes used are isolates from human intestinal samples.
  • the strains are thus part of the normal human intestinal bacterial flora.
  • the introduction of these organisms corrects a deficiency that exists in some humans.
  • Enrichment of the contents of the intestines with one or more species of oxalate- degrading bacteria causes a reduction of oxalate in the intestinal contents.
  • Some of the bacteria carry out oxalate degradation at or near the site of absorption (herein referred to as "locally"). The activity of the bacteria decreases the level of absorption of dietary oxalate.
  • the invention provides a pharmaceutical composition comprising a lysate/enzyme preparation of Oxalobacter bacterium and Oxalobacter enzymes, whereby, the composition is formulated to prevent degradation in acidic conditions.
  • the composition comprises a lysate of substrate-specific Oxalobacter bacteria and oxalate-degrading enzymes.
  • the oxalate-degrading enzymes are preferably formyl-CoA transferase and oxalyl CoA decarboxylase.
  • the lysate/enzyme composition locally stimulates the active transport systems involved in colonic oxalate secretion.
  • the lysate/enzyme composition's intraluminal oxalate-degradative capacity serves to sustain an outwardly directed concentration gradient such that transmural passive movement of oxalate from the blood into the lumen is also enhanced. Together, these independent actions of the Oxalobacter lysate/enzyme preparation can optimally promote enteric elimination of oxalate.
  • Pharmaceutical compositions for the introduction of oxalate degrading bacteria and/or enzymes, lysate/enzyme preparations into the intestine include bacteria, bacterial lysates and/or enzymes that have been lyophilized or frozen in liquid or paste form and encapsulated in a gel capsule or other enteric protection.
  • a non-exhaustive exemplary list of such animals includes mammals such as mice, rats, rabbits, goats, sheep, pigs, horses, cattle, dogs, cats, and primates such as monkeys, apes, and human beings, as well as laboratory and zoological animals.
  • mammals such as mice, rats, rabbits, goats, sheep, pigs, horses, cattle, dogs, cats, and primates such as monkeys, apes, and human beings, as well as laboratory and zoological animals.
  • Those animal subjects known to suffer from hyperoxaluria and development of subsequent CRF are preferred for use in the invention.
  • human patients suffering from primary hyperoxaluria and CRF are suitable animal subjects for use in the invention.
  • the subjects used were rats. Nonetheless, by adapting the methods taught herein to other methods known in medicine or veterinary science (e.g., adjusting doses of administered substances according to the weight of the subject animal), the compositions utilized in the invention can be readily optimized for use in other subjects.
  • compositions and Administration to a Subject may be administered to animals including human beings in any suitable formulation.
  • an Oxalobacter composition e.g., Oxalobacter cells, Oxalobacter enzyme(s), nucleic acid encoding one or more Oxalobacter enzymes
  • an Oxalobacter composition may be formulated in pharmaceutically acceptable carriers or diluents such as physiological saline or a buffered salt solution.
  • Suitable carriers and diluents can be selected on the basis of mode and route of administration and standard pharmaceutical practice.
  • a description of exemplary pharmaceutically acceptable carriers and diluents, as well as pharmaceutical formulations can be found in Remington's Pharmaceutical Sciences, a standard text in this field, and in USP/NF.
  • compositions of the invention may be administered to animals by any conventional technique. Typically, such administration will be oral. However, parenteral administration of the bacteria through suppositories (e.g., intra-anal introduction) or the cell- free enzymes (e.g., intraperitoneal introduction) is also -within the invention.
  • the compositions may also be admimstered directly to a target tissue (e.g., the kidneys) by, for example, direct injection into, or surgical delivery to, an internal or external target tissue.
  • the compositions can be delivered as capsules or microcapsules designed to protect the material from adverse effects of acid stomach. One or more of several enteric protective coating methods can be used.
  • One embodiment of the present invention involves procedures for selection, preparation and administration of the appropriate oxalate-degrading bacteria, bacterial lysate/enzyme preparations to a diversity of subjects. Prominently, but not exclusively, these are persons or animals which do not harbor these bacteria in their intestines. These non- colonized or weakly-colonized persons or animals are identified using tests that allow for rapid and definitive detection of Oxalobacter even when the organisms are at relatively low concentrations in mixed bacterial populations such as are found in intestinal contents.
  • the methods of the subject invention can also be used to treat individuals or animals whose oxalate-degrading bacteria have been depleted due to, for example, antibiotic treatment or in post-operative situations.
  • the methods of the subject invention can also be used to treat individuals or animals who have colonies of oxalate-degrading bacteria but who still have unhealthy levels of oxalate due to, for example, oxalate susceptibility and/or excessive production of endogenous oxalate.
  • Use of pharmaceutically acceptable carriers to formulate the compounds herein disclosed for the practice of the invention into dosages suitable for systemic administration is within the scope of the invention.
  • Suitable lipophilic sol ents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes.
  • Aqueous injection suspensions may contain substances which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran.
  • the suspension may also contain suitable stabilizers or agents which increase the solubility of the compounds to allow for the preparation of highly concentrated solutions.
  • Pharmaceutical preparations for oral use can be obtained by combining the active compounds with solid excipient, optionally grinding a resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries, if desired, to obtain tablets or dragee cores.
  • Suitable excipients are, in particular, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl- cellulose, sodium carboxy-methylcellulose, and or polyvinyl pyrrolidone (PNP).
  • disintegrating agents may be added, such as the cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate.
  • Dragee cores are provided with suitable coating.
  • concentrated sugar solutions may be used, which may optionally contain gum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures.
  • Dyestuffs or pigments may be added to the tablets or dragee coatings for identification or to characterize different combinations of active compound doses.
  • Pharmaceutical preparations which can be used orally include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol.
  • Toxicity and therapeutic efficacy of the compositions utilized in methods of the invention can be determined by standard pharmaceutical procedures, using either cells in culture or experimental animals to determine the LD 50 (the dose lethal to 50% of the population) and the ED 5 o (the dose therapeutically effective in 50% of the population).
  • the dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD 50 /ED 50 .
  • Those compositions that exhibit large therapeutic indices are preferred. While those that exhibit toxic side effects may be used, care should be taken to design a delivery system that minimizes the potential damage of such side effects.
  • the dosage of preferred compositions lies preferably within a range that includes an ED 50 with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized.
  • dosage for any one animal depends on many factors, including the subject's size, body surface area, age, the particular composition to be administered, time and route of administration, general health, and other drugs being administered concurrently. It is expected that an appropriate dosage for oral administration of encapsulated Oxalobacter cells would be in the range of about 1 x 10 7 cells/kg body weight.
  • the methods and compositions of the invention are used to treat patients, preferably humans, with high urinary or serum oxalate levels or hyperoxaluria or who are at risk of high urinary or serum oxalate levels or hyperoxaluria.
  • the Oxalobacter lysates and Oxalobacter enzymes are in a ratio of 5:1 (v/v) up to a ratio of 50:1 (v/v).
  • the method further provides administration of oxalate- degrading enzymes, such as, formyl-CoA transferase and oxalyl CoA decarboxylase.
  • the enzymes can be administered before, during and/or after administration of the composition comprising Oxalobacter lysates and Oxalobacter enzymes.
  • the composition comprising Oxalobacter lysates and Oxalobacter enzymes provide an oxalate degrading capacity of about 50 to about 900mg oxalate/day.
  • Each dose comprises about 1. Omg to about 20 mg of active enzyme component with an oxalate degrading capacity between about 0.1 ⁇ mole to about 100.0 ⁇ mole oxalate/ min/mg protein.
  • Methods for identification and/or quantitation of lysate/enzyme preparations can be obtained by methods well-known in the art. These include, without limitation immunoassays, gels, spectrometry, and the like. Preparation and u.se of the lysate/enzymes are described in detail in the Examples which follow. Methods for mass-scale and rapid quantitation are described in U.S. Patent No.: 6,852,544, incorporated herein by reference.
  • U.S. Patent No.: 6,852,544 provides analytical reagents and mass spectrometry-based methods using these reagents for the rapid, and quantitative analysis of proteins or protein function in mixtures of proteins.
  • the analytical method can be used for qualitative and particularly for quantitative analysis of global protein expression profiles in cells and tissues.
  • the method can also be employed to screen for and identify proteins whose expression levels in cells, tissue or biological fluids are affected by a stimulus (e.g., administration of the compositions described herein), by a change in environment (e.g., nutrient level, temperature, passage of time) or by a change in condition or cell state (e.g., disease state, malignancy, site-directed mutation, gene knockouts) of the cell, tissue or organism from which the sample originated.
  • a stimulus e.g., administration of the compositions described herein
  • a change in environment e.g., nutrient level, temperature, passage of time
  • a change in condition or cell state e.g., disease state, malignancy, site-directed mutation, gene knockouts
  • the proteins identified in such a screen can function as molecules for the changed state. For example, comparisons of protein expression profiles of nomial and renal disease cells can result in the identification of proteins whose presence or absence is characteristic and diagnostic of the renal disease.
  • a sample i.e. Oxalobacter lysate an ⁇ l/or enzyme preparation
  • size exclusion chromatography For a biological sample wherein the amount of sample available is small, preferably a size selection spin column is used.
  • the first fraction that is eluted from the column (“fraction 1") has the highest percentage of high molecular weight proteins; fraction 2 has a lower percentage of high molecular weight proteins; fraction 3 has even a lower percentage of high molecular weight proteins; fraction 4 hias the lowest amount of large proteins; and so on.
  • a sample can be pre-fractionated by anion exchange chromatography.
  • Anion exchange chromatography allows pre-fractionation of the proteins in a sample roughly according to their charge characteristics.
  • a Q anion-exchange resin can be used (e.g., Q HyperD F, Biosepra), and a sample can be sequentially eluted with eluants having different pH's.
  • Anion exchange chromatography allows separation of molecules in a sample that are more negatively charged from other types of molecules.
  • a sample can be pre-fractionated by heparin chromatography. Heparin chromatography allows pre-fractiona-tion of the molecules in a sample also on the basis of affinity interaction with heparin and charge characteristics.
  • Heparin a sulfated mucopolysaccharide
  • a sample can be sequentially eluted with eluants having different pH's or salt concentrations. Molecules eluted with an eluant having a low p ⁇ are more likely to be weakly positively charged. Molecules eluted with an eluant havnng a high pH are more likely to be strongly positively charged.
  • heparin chromatograpt y also reduces the complexity of a sample and separates molecules according to their binding characteristics.
  • a sample can be pre-fractionated by isolating proteins that have a specific characteristic, e.g.
  • a blood, or serum sample can be fractionated by passing the sample over a lectin chromatography column (which has a high affinity for sugars). Glycosylated proteins will bind to the lectin column and non- glycosylated proteins will pass through the flow through. Glycosylated proteins are then eluted from the lectin column with an eluant containing a sugar, e.g., N-acetyl-glucosamine and are available for further analysis.
  • a sample can be fractionated using a sequential extraction protocol.
  • a multi-well plate/system comprising different adsorbents at its bottom can be used.
  • sequential extraction can be performed on a probe adapted for use in a gas phase ion spectrometer, wherein the probe surface comprises adsorbents for binding molecules.
  • the sample is applied to a first adsorbent on the probe, which is subsequently washed with an eluant. Molecules that do not bind to the first adsorbent are removed with an eluant.
  • the molecules that are in the fraction can be applied to a second adsorbent on the probe, and so forth.
  • the advantage of performing sequential extraction on a gas phase ion spectrometer probe is that molecules that bind to various adsorbents at every stage of the sequential extraction protocol can be analyzed directly using a gas phase ion spectrometer.
  • molecules in a sample can be separated by high- resolution electrophoresis, e.g., one or two-dimensional gel electrophoresis.
  • a fraction containing an enzyme can be isolated and further analyzed by gas phase ion spectrometry.
  • two-dimensional gel electrophoresis is used to generate two-dimensional array of spots of molecules, including one or more molecules. See, e.g., Jungblut and Thiede, Mass Spectr. Rev. 16:145-162 (1997).
  • the two-dimensional gel electrophoresis can be performed using methods known in the art. See, e.g., Deutscher ed., Methods In Enzymology vol. 182.
  • molecules in a sample are separated by, e.g., isoelectric focusing, during which molecules in a sample are separated in a pH gradient until they reach a spot where their net charge is zero (i.e., isoelectric point).
  • This first separation step results in one-dimensional array of molecules.
  • the molecules in one dimensional array are further separated using a technique generally distinct from that used in the first separation step.
  • molecules separated by isoelectric focusing are further separated using a polyacrylamide gel, such as polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS- PAGE).
  • SDS-PAGE gel allows further separation based on molecular mass of molecules.
  • two-dimensional gel electrophoresis can separate chemically different molecules in the molecular mass range from 1000-200,000 Da within complex mixtures.
  • Molecules in the two-dimensional array can be detected ixsing any suitable methods known in the art.
  • molecules in a gel can be labeled or stained (e.g., Coomassie Blue or silver staining).
  • the spot can be further analyzed by densitometric analysis or gas phase ion spectrometry.
  • spots can be excised from the gel and analyzed by gas phase ion spectrometry.
  • the gel containing molecules can be transferred to an inert membrane by applying an electric field.
  • a spot on the membrane that approximately corresponds to the molecular weight of, for example, an enzyme can be analyzed by gas phase ion spectrometry.
  • the spots can be analyzed using any suitable techniques, such as MAXDI or SELDI.
  • HPLC high performance liquid chromatography
  • Molecules in a sample are separated by injecting an aliquot of the sample onto the column. Different molecules in the mixture pass through the column at different rates due to differences in their partitioning behavior between the mobile liquid phase and the stationary phase. A fraction that corresponds to the molecular weight and/or physical properties of one or more molecules can be collected. The fraction can then be analyzed by gas phase ion spectrometry to detect molecules.
  • a molecule can be modified before analysis to improve its resolution or to determine its identity.
  • the molecules maybe subject to proteolytic digestion before analysis. Any protease can be used. Proteases, such as trypsin, that are likely to cleave the molecules into a discrete number of fragments are particularly useful.
  • the identity of the molecules can be further determined by matching the physical and ch-emical characteristics of the modified molecules in a protein database (e.g., SwissProt).
  • a protein database e.g., SwissProt
  • molecules in a sample are typically captured on a substrate for detection.
  • Traditional substrates include antibody-coated 96-well plates or nitrocellulose membranes that are subsequently probed for the presence of proteins.
  • the molecules are identified using immunoassays as described above.
  • preferred methods also include the use of biochips.
  • the biochips are protein biochips for capture and detection of proteins. Many protein biochips are described in the art.
  • protein biochips produced by Packard BioScience Company (Meriden CT), Zyomyx (Hayward, CA) and Phylos (Lexington, MA).
  • protein biochips comprise a substrate having a surface.
  • a capture reagent or adsorbent is attached to the surface of the substrate.
  • the surface comprises a plurality of addressable locations, each of which location has the capture reagent bound there.
  • the capture reagent can be a biological molecule, such as a polypeptide or a nucleic acid, which captures other molecules in a specific manner.
  • the capture reagent can be a chromatographic material, such as an anion exchange material or a hydrophilic materiaJ.
  • a substrate or a probe comprising molecules is introduced into an inlet system.
  • the molecules are desorbed and ionized into the gas phase by laser from the ionization source.
  • the ions generated are collected by an ion optic assembly, and then in a time-of-flight mass analyzer, ions are accelerated through a short high voltage field and let drift into a high vacuum chamber. At the far end of the high vacuum chamber, the accelerated ions strike a sensitive detector surface at a different time. Since the time-of- flight is a function of the mass of the ions, the elapsed time between ion formation and ion detector impact can be used to identify the presence or absence of molecules of specific mass to charge ratio.
  • Exemplary energy absorbing molecules include cinnamic acid derivatives, sinapinic acid (“SPA”), cyano hydroxy cinnamic acid (“CHCA”) and dihydroxybenzoic acid. Other suitable energy absorbing molecules are known to those skilled in this art.
  • the matrix dries, forming crystals that encapsulate the analyte molecules. Then the analyte molecules are detected by laser desorption/ionization mass spectrometry.
  • MALDI-MS is useful for detecting the molecules of this invention if the complexity of a sample has been substantially reduced using the preparation methods described above.
  • SELDI is described, for example, in: United States Patent 5,719,060 ("Method and Apparatus for Desorption and Ionization of Analytes," Hutchens and Yip, February 17, 1998,) United States Patent 6,225,047 ("Use of Retentate Chromatography to Generate Difference Maps," Hutchens and Yip, May 1, 2001) and Weinberger et al, "Time-of-flight mass spectrometry," in Encyclopedia of Analytical Chemistry, R.A. Meyers, ed., pp 11915-11918 John Wiley & Sons Chichesher, 2000. Molecules on the substrate surface can be desorbed and ionized using gas phase ion spectrometry.
  • gas phase ion spectrometers can be used as long as it allows molecules on the substrate to be resolved.
  • gas phase ion spectrometers allow quantitation of molecules.
  • a gas phase ion spectrometer is a mass spectrometer.
  • a substrate or a probe comprising molecules on its surface is introduced into an inlet system of the mass spectrometer.
  • the molecules are then desorbed by a desorption source such as a laser, fast atom bombardment, high energy plasma, electrospray ionization, thermospray ionization, liquid secondary ion MS, field desorption, etc.
  • the generated desorbed, volatilized species consist of preformed ions or neutrals which are ionized as a direct consequence of the desorption event.
  • Generated ions are collected by an ion optic assembly, and then a mass analyzer disperses and analyzes the passing ions.
  • the ions exiting the mass analyzer are detected by a detector.
  • the detector then translates information of the detected ions into mass-to-charge ratios. Detection of the presence of molecules or other substances will typically involve detection of signal intensity. This, in turn, can reflect the quantity and character of molecules bound to the substrate.
  • an immunoassay can be used to detect and analyze molecules in a sample. This method comprises: (a) providing an antibody that specifically binds to a molecule of interest; (b) contacting a sample with the antibody; and (c) detecting the presence of a complex of the antibody bound to the molecule in the sample. To prepare an antibody that specifically binds to an unknown molecule, purified molecules or their nucleic acid sequences can be used.
  • Nucleic acid and amino acid sequences for molecules can be obtained by further characterization of these molecules.
  • each marker can be peptide mapped with a number of enzymes (e.g., trypsin, N8 protease, etc.).
  • the molecular weights of digestion fragments from each marker can be used to search the databases, such as SwissProt database, for sequences that will match the molecular weights of digestion fragments generated by various enzymes.
  • the nucleic acid and amino acid sequences of other molecules can be identified if these molecules are known proteins in the databases.
  • the proteins can be sequenced using protein ladder sequencing.
  • Protein ladders can be generated by, for example, fragmenting the molecules and subjecting fragments to enzymatic digestion or other methods that sequentially remove a single amino acid from the end of the fragment. Methods of preparing protein ladders are described, for example, in International Publication WO 93/24834 (Chait et al.) and United States Patent 5,792,664 (Chait et al.). The ladder is then analyzed by mass spectrometry. The difference in the masses of the ladder fragments identify the amino acid removed from the end of the molecule. If the molecules are not known proteins in the databases, nucleic acid and amino acid sequences can be determined with knowledge of even a portion of the amino acid sequence of the molecule.
  • degenerate probes can be made based on the ⁇ -terminal amino acid sequence of, for example, the active molecule in the lysate. These probes can then be used to screen a genomic or cD ⁇ A library created from a sample from which a molecule was initially detected. The positive clones can be identified, amplified, and their recombinant
  • D ⁇ A sequences can be subcloned using techniques which are well known. See, e.g., Current Protocols for Molecular Biology (Ausubel et al., Green Publishing Assoc. and Wiley- Interscience 1989) and Molecular Cloning: A Laboratory Manual, 3rd Ed. (Sambrook et al., Cold Spring Harbor Laboratory, ⁇ Y 2001). Using the purified molecules or their nucleic acid sequences, antibodies that specifically bind to them can be prepared using any suitable methods known in the art. See, e.g., Coligan, Current Protocols in Immunology (1991); Harlow & Lane, Antibodies: A Laboratory Manual (1988); Goding, Monoclonal Antibodies: Principles and Practice (2d ed.
  • Such techniques include, but are not limited to, antibody preparation by selection of antibodies from libraries of recombinant antibodies in phage or similar vectors, as well as preparation of polyclonal and monoclonal antibodies by immunizing rabbits or mice (see, e.g., Huse et al., Science 246:1275-1281 (1989); Ward et al., Nature 341:544-546 (1989)).
  • enzymes or other active molecules in a lysate preparation can be detected and/or quantified using any of suitable immunological binding assays known in the art (see, e.g., U.S. Patent Nos.
  • Useful assays include, for example, an enzyme immune assay (EIA) such as enzyme-linked immunosorbent assay (ELISA), a radioimmune assay (RIA), a Western blot assay, or a slot blot assay.
  • EIA enzyme immune assay
  • ELISA enzyme-linked immunosorbent assay
  • RIA radioimmune assay
  • Western blot assay or a slot blot assay.
  • the antibody can be fixed to a solid support to facilitate washing and subsequent isolation of the complex, prior to contacting the antibody with a sample.
  • solid supports include glass or plastic in the form of, e.g., a microtiter plate, a stick, a bead, or a microbead.
  • Antibodies can also be attached to a probe substrate or ProteinChip® array described above.
  • the sample is preferably a lysate of Oxalobacter.
  • the sample can be diluted with a suitable eluant before contacting the sample to the antibody.
  • the mixture is washed and the antibody- antigen complex formed can be detected. This can be accomplished by incubating the washed mixture with a detection reagent.
  • This detection reagent may be, e.g., a second antibody which is labeled with a detectable label.
  • Exemplary detectable labels include magnetic beads (e.g., DYNABEADSTM), fluorescent dyes, radiolabels, enzymes (e.g., horse radish peroxide, alkaline phosphatase and others commonly used in an ELISA), and colorimetric labels such as colloidal gold or colored glass or plastic beads.
  • the active molecules in an Oxalobacter lysate can be detected using an indirect assay, wherein, for example, a second, labeled antibody is used to detect bound active molecule-specific antibody, and/or in a competition or inhibition assay wherein, for example, a monoclonal antibody which binds to a distinct epitope of the molecule is incubated simultaneously with the mixture.
  • incubation and/or washing steps may be required after each combination of reagents. Incubation steps can vary from about 5 seconds to several hours, preferably from about 5 minutes to about 24 hours. However, the incubation time will depend upon the assay format, molecule, volume of solution, concentrations and the like. Usually the assays will be carried out at ambient temperature, although they can be conducted over a range of temperatures, such as 10°C to 40°C. Immunoassays can be used to determine presence or absence of a protein in a sample as well as the quantity of a protein in a sample. First, a test amount of a protein in a sample can be detected using the immunoassay methods described above.
  • a protein If a protein is present in the sample, it will form an antibody-protein complex with an antibody that specifically binds the protein under suitable incubation conditions described above.
  • the amount of an antibody-protein complex can be determined by comparing to a standard.
  • a standard can be, e.g., a known compound or another protein known to be present in a sample.
  • the test amount of protein need not be measured in absolute units, as long as the unit of measurement can be compared to a control.
  • the methods for detecting these molecules in a sample have many applications. For example, one or more molecules can be measured to aid in the diagnosis of renal disorders. In another example, the methods for detection of the molecules can be used to monitor responses of a subject to treatment.
  • the methods for detecting molecules can be used to assay for and to identify compounds that modulate expression of these molecules in vivo or in vitro.
  • Data generated by desorption and detection of molecules can be analyzed using any suitable means.
  • data is analyzed with the use of a programmable digital computer.
  • the computer program generally contains a readable medium that stores codes. Certain code can be devoted to memory that includes the location of each feature on a probe, the identity of the adsorbent at that feature and the elution conditions used to wash the adsorbent.
  • the computer also contains code that receives as input, data on the strength of the signal at various molecular masses received from a particular addressable location on the probe.
  • This data can indicate the number of molecules detected, including the strength of the signal generated by each marker.
  • Data analysis can include the steps of determining signal strength (e.g., height of peaks) of a marker detected and removing "outliers" (data deviating from a predetermined statistical distribution).
  • the observed peaks can be normalized, a process whereby the height of each peak relative to some reference is calculated.
  • a reference can be background noise generated by instrument and chemicals (e.g., energy absorbing molecule) which is set as zero in the scale.
  • the signal strength detected for each marker or other biomolecules can be displayed in the form of relative intensities in the scale desired (e.g., 100).
  • a standard e.g., a serum protein
  • the computer can transform the resulting data into various formats for displaying.
  • spectrum view or retentate map a standard spectral view can be displayed, wherein the view depicts the quantity of marker reaching the detector at each particular molecular weight.
  • peak map only the peak height and mass information are retained from the spectrum view, yielding a cleaner image and enabling molecules with nearly identical molecular weights to be more easily seen.
  • each mass from the peak view can be converted into a grayscale image based on the height of each peak, resulting in an appearance similar to bands on electrophoretic gels.
  • 3-D overlays several spectra can be overlaid to study subtle changes in relative peak heights.
  • difference map view two or more spectra can be compared, conveniently highlighting unique molecules and molecules which are up- or down-regulated between samples. Protein profiles (spectra) from any two samples may be compared visually.
  • Oxalobacter and preparation of Oxalobacter-derived products Growth of bacterial strains was conducted in large fermenter vessels.
  • the strains selected to represent the diversity within the genus Oxalobacter include three from Group I: strain OxB, the type strain; strain OxWR isolated from a wild rat; and strain HC-1 from a human fecal sample.
  • the two strains from Group II are: strain OxCR from a laboratory rat and strain BA-1 from a human fecal sample.
  • the various lysate preparations made from each of these strains used at three different concentrations in separate series of flux experiments (final protein concentration 0.54 mg/ml of mucosal bathing solution was used in experiments).
  • the bacteria are grown in either 100 liter or 15 liter fermenter vessels and harvested using a continuous flow centrifuge. The oxalate degradative capacity of the cell preparation is determined as described previously (Allison, M.
  • the lysed cells are centrifuged at 15,000 g for 20 minutes to remove cell debris.
  • the lysate is further clarified by ultrafiltration through 0.45 ⁇ filter.
  • This final lysate preparation is immediately stored at -80°C.
  • an aliquot is subjected to lyopholization.
  • Each batch of the lysate is tested for protein concentration, protein profile by SDS-PAGE chromatography and for its oxalate degrading activity. Enzyme activity is measured before the lysate is frozen and again when it is thawed out for testing in the flux experiments. (iii) Determination of oxalate degrading activity of the O.
  • Oxalobacter lysate as the source of formyl-CoA transferase and oxalyl-CoA decarboxylase. Initial rates of the reaction are recorded and calculated by the increase in absorbance at 340 nm using the extinction coefficient for NAD.
  • a cell lysate is prepared from one of the strains of Oxalobacter.
  • the choice of strain is one which produces optimal colonic oxalate secretion and the lysate with known oxalate degrading capacity (measured in an in vitro assay) are placed in the capsules with oxalyl CoA, TPP and MgCl 2 as co-factors.
  • the minicapsules (size 9) especially designed for rat studies (Torpac Inc., NJ. Torpac Inc., also sells a capsule filling and feeding device that has been successfully used for prior studies) is coated with Eudragit L 100-55 (Huls America Inc., NJ).
  • Eudragit L 100-55 provides an enteric coating to the capsules that safely takes the supplement through the highly acidic gastric contents. It was demonstrated, in in vitro studies, that Eudragit coated capsules showed no dissolution at 37° C at pH ⁇ 6.0.
  • the capsules provide an oxalate degrading capacity of approximately 200-500mg oxalate /day.
  • Each capsule contains about 1.5 - 2 mg of active enzyme component with an oxalate degrading capacity between 0.8 - 1.0 ⁇ mole oxalate/ min/mg protein.
  • the capsules are placed at the end of an intragastic needle and administered to the rats by intragastric intubation at the beginning and the end of each day, for a 7-day period.
  • Oxalate Flux Studies Oxalate flux measurements using in vitro intestinal tissue preparations are routine in applicant's laboratory. Briefly, the distal colon is removed from euthanized rats. The colonic segments are rinsed in a standard buffered saline and the sub-mucosal connective tissues and muscle layers are removed using blunt dissection. Sheets of colonic tissue are mounted in Ussing chambers with an exposed area of 0.64 cm 2 . Standard saline solutions bathe either face of the tissue and these are maintained at 37° C and circulated by bubbling with a gas mixture, 95% O 2 / 5% CO 2 .
  • Transepithelial fluxes of 14 C-oxalate are measured under short-circuit conditions with an automatic voltage clamping device, (VCC600, Physiologic Instruments, San Diego, CA) as described previously (Freel, R. W., M. Hatch, D. L. Earnest, and A. M. Goldner. Oxalate transport across the isolated rat colon. Biochim Biophys Ada 600: 838-43, 1980; Hatch, M., R. W. Freel, andN. D. Vaziri. Am Soc Nephrol 5: 1339-43, 1994).
  • VCC600 automatic voltage clamping device
  • Kidneys and Evaluation of Renal Damage Animals are euthanized with an intraperitoneal injection of sodium pentobarbital. The kidneys are removed and fixed in 10% neutral buffered formalin, trimmed, processed, and embedded in paraffin. Two sections from each kidney are stained with hematoxylin and eosin and examined under polarized light. The presence of CaOx crystals are scored on a basis of 0-4+ . The rest of the kidney tissue is saved for the determination of other indicators of renal injury as follows:
  • Urine and Plasma Oxalate Determination The measurement of oxalate in urine and plasma specimens is routine in our laboratory. The enzymatic, sensitive, and specific assay procedure currently being used was developed by Hatch et al. (Hatch, M. Spectrophotometric determination of oxalate in whole blood. Clin. Chim. Acta 193: 199, 1990; Hatch, M., E. Bourke, and J. Costello. New enzymatic method for serum oxalate determination. Clin. Chem. 23: 76-80, 1977.) and it has been employed to determine oxalate concentrations in numerous studies (using humans and animals). Calcium and creatinine is determined in the urine and plasma samples using the Sigma kit assays #587A and #555A, respectively (Sigma Chemical Co., St. Louis, MO).
  • composition of a particular diet is referred to by way of the gram amount of oxalate or Ca 2+ present in that diet per 100 gm of chow i.e. on a percent basis.
  • the low Ca 2+ diet contained 0.01% Ca 2+ (Product # TD 99354) and the high Ca 2+ diet contained 1.2% Ca 2+ (Product # TD 99355).
  • the oxalate content of the diet was altered by simply quantitatively reducing the amount of oxalate salt added to the powder.
  • the magnitude and direction of the net flux of oxalate (j ⁇ ) was determined by calculating the difference between two measured unidirectional fluxes (mucosal to serosal, J°* and serosal to mucosal, J°*).
  • the experimental design consisted of a 45 min. period (Period I) during which time fluxes and electrical parameters were measured at 15 min intervals.
  • Tissue conductance G ⁇ 5 mS-cm "2
  • Urinary oxalate was reduced 50% (102 +/- 11 to 57 +/- 8 ⁇ mol/24 hrs), and Oxalobacter lysate treatment induced local oxalate secretion in the distal colon. Coupled together, these independent and separate actions of O. formigenes and/or lysate/enzyme preparations can promote an enhanced enteric elimination of oxalate.
  • the administration of O. formigenes, or products thereof can be used to enhance the extra-renal elimination of oxalate from the circulatory system via the intestines, thereby reducing the burden of oxalate in the kidney. This extra-renal elimination is actually enhanced when CRF is present.
  • Example 2 Effectiveness of the enteric lysate/enzyme preparation The proposed mechanism underlying the effectiveness of the enteric lysate/ enzymes preparation is based upon two actions: First, it locally stimulates the active transport systems involved in colonic oxalate secretion.
  • Example 4 Induction of Colonic Oxalate Secretion It was hypothesized that Oxalobacter sp. possess a strategic ability to optimize substrate availability within the intestinal lumen. The effects of select bacterial preparations on epithelial oxalate transport were directly tested in a series of in vitro flux experiments. The following preparations were used: preparations of OxWR (wild rat strain of Oxalobacter); a preparation of washed, whole Oxalobacter cells, a preparation of washed Oxalobacter cell membrane fragments, and a preparation of Oxalobacter cell lysate. Distal colonic tissues removed from rats, not colonized and fed the standard Purina chow 5001 diet, were used in these experiments.
  • OxWR wild rat strain of Oxalobacter
  • Oxalate fluxes were measured before and after the addition of the lysate to the solution bathing the mucosal side of colonic tissues.
  • Example 5 The Oxalate-induced Chronic Renal Failure (CRF) Rat Model
  • CRF Chronic Renal Failure Rat Model
  • the aim of the study was to determine the effectiveness of an oxalate-degrading enzyme supplementation therapy on reducing urinary oxalate in renal failure associated with chronic hyperoxaluria.
  • the interest in " using this particular model was two-fold: First, the distal colon supports a net secretory fltrx of oxalate that is induced by CRF. Second, persistent hyperoxaluria, due to an endogenous overproduction of oxalate is a feature of this animal model. It was determined that 4 weeks of treating unilateral nephrectomized rats with 0.75% ethylene glycol resulted in a two-fold increase in plasma creatinine.
  • Example 6 Enzyme supplementation therapy in CRF rats
  • oxalate-degrading enzyme supplementation therapy was administered 0.75% ethylene glycol in their drinking water after a recovery period of one week.
  • the results are especially significant because they show that the balance " between renal and enteric excretion of endogenously- derived oxalate, in contrast to food oxalate present in the luminal environment, can be manipulated.
  • the oxalate burden in this animal model was derived from ethylene glycol metabolism and the food supplied to these rats was not supplemented with oxalate.
  • the mechanism underlying the effectiveness of the enteric lysate/ enzymes preparation is based upon two actions: First, it locally stimulates the active transport systems involved in colonic oxalate secretion. Second, its intraluminal oxalate- degradative capacity serves to sustain an outwardly directed concentration gradient such that transmural passive movement of oxalate from the blood into the lumen is also enhanced.
  • the results obtained in the in vitro experiments using the bacterial lysate (presented above, Figure 6) are consistent with both notions. Together, these independent actions of the Oxalobacter lysate/enzyme preparation can optimally promote enteric elimination of oxalate. In conclusion, it appears prudent to direct the Phase II Specific Aims towards the development of a supplemental lysate/ enzymes therapy for the control of hyperoxaluric conditions.

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Abstract

La présente invention concerne des compositions et des méthodes d'amélioration de la vitesse de transport de l'oxalate d'un site parentéral à la lumière intestinale chez un animal. Les compositions et les méthodes sont utiles pour traiter un animal atteint d'une insuffisance rénale. Lorsqu'on augmente le nombre de bactéries Oxalobacter ou d'enzymes Oxalobacter chez l'animal, l'oxalate est transporté à la lumière intestinale et dégradé dans ladite lumière intestinale, ce qui réduit ainsi la charge d'excrétion de l'oxalate effectuée par les reins.
PCT/US2005/010227 2004-03-26 2005-03-25 Modulation du transport d'oxalate WO2005097176A2 (fr)

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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2015002604A1 (fr) 2013-07-05 2015-01-08 Oxthera Intellectual Property Ab Sécrétagogues dérivés d'oxalobacter formigenes
WO2020018799A1 (fr) 2018-07-18 2020-01-23 The University Of Chicago Compositions comprenant des peptides dérivés de sel1 et méthodes de traitement/prévention de taux d'oxalate en excès et pathologies/maladies associés à ceux-ci
WO2020071994A1 (fr) * 2018-10-04 2020-04-09 Oxthera Intellectual Property Ab Nouvelle utilisation médicale de bactéries de réduction de l'oxalate
US10765709B2 (en) 2016-06-13 2020-09-08 Oxthera Intellectual Property Compositions and methods for the treatment or prevention of oxalate-related disorders
CN114645005A (zh) * 2022-05-18 2022-06-21 中国科学院地理科学与资源研究所 一种假单胞菌及其应用

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20020102238A1 (en) * 1997-05-23 2002-08-01 Allison Milton J. Materials and methods for treating or preventing oxalate-related disease

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20020102238A1 (en) * 1997-05-23 2002-08-01 Allison Milton J. Materials and methods for treating or preventing oxalate-related disease

Cited By (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2015002604A1 (fr) 2013-07-05 2015-01-08 Oxthera Intellectual Property Ab Sécrétagogues dérivés d'oxalobacter formigenes
WO2015002588A1 (fr) * 2013-07-05 2015-01-08 Oxthera Intellectual Property Ab Sécrétagogues dérivés d'oxalobacter formigenes
CN105358172A (zh) * 2013-07-05 2016-02-24 奥克斯泰拉知识产权公司 源自产甲酸草酸杆菌的促分泌素
US10125176B2 (en) 2013-07-05 2018-11-13 Oxthera Intellectual Property Ab Secretagogues derived from oxalobacter formigenes
EP3865148A1 (fr) 2013-07-05 2021-08-18 OxThera Intellectual Property AB Sécrétagogues dérivés d'oxalobacter formigenes
US10988510B2 (en) 2013-07-05 2021-04-27 Oxthera Intellectual Property Ab Secretagogues derived from oxalobacter formigenes
US10765709B2 (en) 2016-06-13 2020-09-08 Oxthera Intellectual Property Compositions and methods for the treatment or prevention of oxalate-related disorders
WO2020018799A1 (fr) 2018-07-18 2020-01-23 The University Of Chicago Compositions comprenant des peptides dérivés de sel1 et méthodes de traitement/prévention de taux d'oxalate en excès et pathologies/maladies associés à ceux-ci
EP3836949A4 (fr) * 2018-07-18 2022-03-09 The University of Chicago Compositions comprenant des peptides dérivés de sel1 et méthodes de traitement/prévention de taux d'oxalate en excès et pathologies/maladies associés à ceux-ci
US12016899B2 (en) 2018-07-18 2024-06-25 The University Of Chicago Compositions comprising Sel1-derived peptides and methods of treatment/prevention of excess oxalate levels and associated conditions/diseases therewith
WO2020071994A1 (fr) * 2018-10-04 2020-04-09 Oxthera Intellectual Property Ab Nouvelle utilisation médicale de bactéries de réduction de l'oxalate
EP3860628A4 (fr) * 2018-10-04 2022-08-17 OxThera Intellectual Property AB Nouvelle utilisation médicale de bactéries de réduction de l'oxalate
CN114645005A (zh) * 2022-05-18 2022-06-21 中国科学院地理科学与资源研究所 一种假单胞菌及其应用

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